Molecular recognition of human eosinophil-derived neurotoxin (RNase 2) by placental ribonuclease inhibitor

Placental ribonuclease inhibitor (RI) binds diverse mammalian RNases with dissociation constants that are in the femtomolar range. Previous studies on the complexes of RI with RNase A and angiogenin revealed that RI utilises largely distinctive interactions to achieve high affinity for these two ligands. Here we report a 2.0 angstroms resolution crystal structure of RI in complex with a third ligand, eosinophil-derived neurotoxin (EDN), and a mutational analysis based on this structure. The RI-EDN interface is more extensive than those of the other two complexes and contains a considerably larger set of interactions. Few of the contacts present in the RI-angiogenin complex are replicated; the correspondence to the RI-RNase A complex is somewhat greater, but still modest. The energetic contributions of various interface regions differ strikingly from those in the earlier complexes. These findings provide insight into the structural basis for the unusual combination of high avidity and relaxed stringency that RI displays.     

Alcohol is a potent stimulant of immature neuronal networks: implications for fetal alcohol spectrum disorder

Ethanol consumption during development affects the maturation of hippocampal circuits by mechanisms that are not fully understood. Ethanol acts as a depressant in the mature CNS and it has been assumed that this also applies to immature neurons. We investigated whether ethanol targets the neuronal network activity that is involved in the refinement of developing hippocampal synapses. This activity appears during the growth spurt period in the form of giant depolarizing potentials (GDPs). GDPs are generated by the excitatory actions of GABA and glutamate via a positive feedback circuit involving pyramidal neurons and interneurons. We found that ethanol potently increases GDP frequency in the CA3 hippocampal region of slices from neonatal rats. It also increased the frequency of GDP-driven Ca2+ transients in pyramidal neurons and increased the frequency of GABA(A) receptor-mediated spontaneous postsynaptic currents in CA3 pyramidal cells and interneurons. The ethanol-induced potentiation of GABAergic activity is probably the result of increased quantal GABA release at interneuronal synapses but not enhanced neuronal excitability. These findings demonstrate that ethanol is a potent stimulant of developing neuronal circuits, which might contribute to the abnormal hippocampal development associated with fetal alcohol syndrome and alcohol-related neurodevelopmental disorders.     

Common binding site for disialyllactose and tri-peptide in C-fragment of tetanus neurotoxin

Clostridial neurotoxins are comprised of botulinum (BoNT) and tetanus (TeNT), which share significant structural and functional similarity. Crystal structures of the binding domain of TeNT complexed with disialyllactose (DiSia) and a tri-peptide Tyr-Glu-Trp (YEW) have been determined to 2.3 and 2.2 A, respectively. Both DiSia and YEW bind in a shallow cleft region on the surface of the molecule in the beta-trefoil domain, interacting with a set of common residues, Asp1147, Asp1214, Asn1216, and Arg1226. DiSia and YEW binding at the same site in tetanus toxin provides a putative site that could be occupied either by a ganglioside moiety or a peptide. Soaking experiments with a mixture of YEW and DiSia show that YEW competes with DiSia, suggesting that YEW can be used to block ganglioside binding. A comparison with the TeNT binding domain in complex with small molecules, BoNT/A and /B, provides insight into the different modes of ganglioside binding.     

The Tetanus Neurotoxin‐Sensitive and Insensitive Routes to and from the Plasma Membrane: Fast and Slow Pathways?

Intracellular membrane trafficking in eukaryotes involves the budding of vesicles from a donor compartment, their translocation, and subsequent fusion with a target membrane. This last step has been shown to involve SNARE proteins, classified into two categories, vesicular (v)-SNAREs and target (t)-SNAREs. It is the pairing of v- and t-SNAREs that is responsible for bringing the lipid bilayers together for membrane fusion. Key to the discovery of SNAREs is the sensitivity of their neuronal synaptic prototypes, which mediate the release of neurotransmitters, to clostridial neurotoxins. In this review, we focus on tetanus neurotoxin-sensitive and tetanus neurotoxin-insensitive v-SNAREs, in particular synaptobrevin and cellubrevin, both tetanus neurotoxin-sensitive and Tetanus neurotoxin-Insensitive Vesicle-Associated Membrane Protein (TI-VAMP, also called VAMP7). The brevins are characterized by an RD sequence in the middle of their SNARE motif whereas TI-VAMP has an RG sequence. These two categories of exocytic v-SNAREs define two important routes to and from the plasma membrane: one sensitive, the other insensitive to tetanus neurotoxin. We also discuss the central role of the endosomal system that could be considered, as already suggested for Rab proteins, as a mosaic of v-SNAREs, thus raising the question of whether or not these two routes can merge, and if so, how and where.     

Dopamine transporter-mediated cytotoxicity of beta-carbolinium derivatives related to Parkinson's disease: relationship to transporter-dependent uptake

Endogenous or exogenous beta-carboline (betaC) derivatives structurally related to the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP(+)) may contribute to dopaminergic neurodegeneration in Parkinson's disease (PD). We addressed the importance of the dopamine transporter (DAT) for selective dopaminergic toxicity by testing the differential cytotoxicity and cellular uptake of 12 betaCs in human embryonic kidney HEK-293 cells ectopically expressing the DAT gene. Cell death was measured using [4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and trypan blue exclusion assays, and uptake by a fluorescence-based uptake assay. All betaCs and MPP(+) showed general cytotoxicity in parental HEK-293 cells after 72 h with half-maximal toxic concentrations (TC(50) values) in the upper micromolar range. Besides MPP(+), only 2[N]-methylated compounds showed enhanced cytotoxicity in DAT expressing HEK-293 cells with 1.3- to 4.5-fold reduction of TC(50) values compared with parental cell line. The rank order of selectivity was: MPP(+) > 2[N],9[N]-dimethyl-harminium > 2[N]-methyl-harminium > 2[N],9[N]-dimethyl-harmanium = 2[N]-methyl-norharmanium > 2[N]-methyl-harmanium > 2[N],9[N]-dimethyl-norharminium. Consistently, only 2[N]-methylated betaCs were transported into the cell through the DAT with up to five times greater K(m) and 12-220 times smaller V(max) values compared with dopamine and MPP(+). There was a weak relation of DAT-mediated selectivity with the affinity of betaCs at the DAT (K(m)), but not with V(max). Our data suggest that DAT-mediated cellular uptake of 2[N]-methylated betaCs represents a potential mechanism for selective toxicity towards dopaminergic neurons and may be relevant for the pathogenesis of Parkinson's disease.     

Botulinum neurotoxin type D enables cytosolic delivery of enzymatically active cargo proteins to neurones via unfolded translocation intermediates

Multi-domain bacterial protein toxins are being explored as potential carriers for targeted delivery of biomolecules. Previous approaches employing isolated receptor binding subunits disallow entry into the cytosol. Strategies in which catalytic domains are replaced with cargo molecules are presumably inefficient due to co-operation of domains during the endosomal translocation step. Here, we characterize a novel transport vehicle in which cargo proteins are attached to the amino terminus of the full-length botulinum neurotoxin type D (BoNT/D). The intrinsic enzymatic activity of the neurotoxin allowed quantification of the efficacy of cargo delivery to the cytosol. Dihydrofolate reductase and BoNT type A (BoNT/A) light chain (LC) were efficiently conveyed into the cytosol, whereas attachment of firefly luciferase or green fluorescent protein drastically reduced the toxicity. Luciferase and BoNT/A LC retained their catalytic activity as evidenced by luciferin conversion or SNAP-25 hydrolysis in the cytosol of synaptosomes, respectively. Conformationally stabilized dihydrofolate reductase as cargo considerably decreased the toxicity indicative for the requirement of partial unfolding of cargo protein and catalytic domain as prerequisite for efficient translocation across the endosomal membrane. Thus, enzymatically inactive clostridial neurotoxins may serve as effective, safe carriers for delivering proteins in functionally active form to the cytosol of neurones.     

Reversible skeletal neuromuscular paralysis induced by different lysophospholipids

Lysophosphatidylcholine rapidly paralyses the neuromuscular junction (NMJ), similarly to snake phospholipase A2 neurotoxins, implicating a lipid hemifusion-pore transition in neuroexocytosis. The mode and kinetics of NMJ paralysis of different lysophospholipids (lysoPLs) in high or low [Mg2+] was investigated. The following order of potency was found: lysophosphatidylcholine>lysophosphatidylethanolamine>lysophosphatidic acid>lysophosphatidylserine>lysophosphatidylglycerol. The latter two lysoPLs closely mimic the profile of paralysis caused by the toxins in high [Mg2+]. This paralysis is fully reversed by albumin washing. These findings provide novel insights on the mode of action of snake neurotoxins and qualify lysoPLs as novel agents to study neuroexocytosis.     

Engineered domain-based assays to identify individual antibodies in oligoclonal combinations targeting the same protein

Quantitation of individual monoclonal antibodies (mAbs) within a combined antibody drug product is required for preclinical and clinical drug development. We have developed two antitoxins, XOMA 3B and XOMA 3E, each consisting of three mAbs that neutralize type B and type E botulinum neurotoxin (BoNT/B and BoNT/E) to treat serotype B and E botulism. To develop mAb-specific binding assays for each antitoxin, we mapped the epitopes of the six mAbs. Each mAb bound an epitope on either the BoNT light chain (LC) or translocation domain (H_N). Epitope mapping data were used to design LC-H_N domains with orthogonal mutations to make them specific for only one mAb in either XOMA 3B or XOMA 3E. Mutant LC-H_N domains were cloned, expressed, and purified from Escherichia coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. Further engineering of domains allowed construction of enzyme-linked immunosorbent assays (ELISAs) that could characterize the integrity, binding affinity, and identity of each of the six mAbs in XOMA 3B and 3E without interference from the three BoNT/A mAbs in XOMA 3AB. Such antigen engineering is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that bind the same protein.     

P19 embryonal carcinoma cells exhibit high sensitivity to botulinum type C and D/C mosaic neurotoxins

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release at peripheral nerve terminals. They are serologically classified from A to G, C/D and D/C mosaic neurotoxins forming further subtypes of serotypes C and D. Cultured primary neurons, as well as neuronal cell lines such as PC12 and Neuro-2a, are often utilized in cell-based experiments on the toxic action of botulinum toxins. However, there are very few reports of the use of neural cell lines for studying BoNTs/C and D. In addition, the differentiated P19 neuronal cell line, which possesses cholinergic properties, has yet to be tested for its susceptibility to BoNTs. Here, the responsiveness of differentiated P19 cells to BoNT/C and BoNT/DC is reported. Both BoNT/C and BoNT/DC were shown to effectively bind to, and be internalized by, neurons derived from P19 cells. Subsequently, the intracellular substrates for BoNT/C and BoNT/DC were cleaved by treatment of the cells with the toxins in a ganglioside-dependent manner. Moreover, P19 neurons exhibited high sensitivity to BoNT/C and BoNT/DC, to the same extent as cultured primary neurons. These findings suggest that differentiated P19 cells possess full sensitivity to BoNT/C and BoNT/DC, thus making them a novel susceptible cell line for research into BoNTs.     

Physicochemical characterization and functional analysis of some snake venom toxin proteins and related non-toxin proteins of other chordates

Snake venom contains a diverse array of proteins and polypeptides. Cytotoxins and short neurotoxins are non-enzymatic polypeptide components of snake venom. The three-dimensional structure of cytotoxin and short neurotoxin resembles a three finger appearance of three-finger protein super family. Different family members of three-finger protein super family are employed in diverse biological functions. In this work we analyzed the cytotoxin, short neurotoxin and related non-toxin proteins of other chordates in terms of functional analysis, amino acid compositional (%) profile, number of amino acids, molecular weight, theoretical isoelectric point (pI), number of positively charged and negatively charged amino acid residues, instability index and grand average of hydropathy with the help of different bioinformatical tools. Among all interesting results, profile of amino acid composition (%) depicts that all sequences contain a conserved cysteine amount but differential amount of different amino acid residues which have a family specific pattern. Involvement in different biological functions is one of the driving forces which contribute the vivid amino acid composition profile of these proteins. Different biological system dependent adaptation gives the birth of enriched bio-molecules. Understanding of physicochemical properties of these proteins will help to generate medicinally important therapeutic molecules for betterment of human lives.